ILK Induces Cardiomyogenesis in the Human Heart

Integrin-linked kinase (ILK) is a widely conserved serine/threonine kinase that regulates diverse signal transduction pathways implicated in cardiac hypertrophy and contractility. In this study we explored whether experimental overexpression of ILK would up-regulate morphogenesis in the human fetal heart.Primary cultures of human fetal myocardial cells (19-22 weeks gestation) yielded scattered aggregates of cardioblasts positive for the early cardiac lineage marker nk × 2.5 and containing nascent sarcomeres. Cardiac cells in colonies uniformly expressed the gap junction protein connexin 43 (C × 43) and displayed a spectrum of differentiation with only a subset of cells exhibiting the late cardiomyogenic marker troponin T (cTnT) and evidence of electrical excitability. Adenovirus-mediated overexpression of ILK potently increased the number of new aggregates of primitive cardioblasts (p<0.001). The number of cardioblast colonies was significantly decreased (p<0.05) when ILK expression was knocked down with ILK targeted siRNA. Interestingly, overexpression of the activation resistant ILK mutant (ILK(R211A)) resulted in much greater increase in the number of new cell aggregates as compared to overexpression of wild-type ILK (ILK(WT)). The cardiomyogenic effects of ILK(R211A) and ILK(WT) were accompanied by concurrent activation of β-catenin (p<0.001) and increase expression of progenitor cell marker islet-1, which was also observed in lysates of transgenic mice with cardiac-specific over-expression of ILK(R211A) and ILK(WT). Finally, endogenous ILK expression was shown to increase in concert with those of cardiomyogenic markers during directed cardiomyogenic differentiation in human embryonic stem cells (hESCs).In the human fetal heart ILK activation is instructive to the specification of mesodermal precursor cells towards a cardiomyogenic lineage. Induction of cardiomyogenesis by ILK overexpression bypasses the requirement of proximal PI3K activation for transduction of growth factor- and β1-integrin-mediated differentiation signals. Altogether, our data indicate that ILK represents a novel regulatory checkpoint during human cardiomyogenesis

The Impairment of ILK Related Angiogenesis Involved in Cardiac Maladaptation after Infarction

Background: Integrin linked kinase (ILK), as an important component of mechanical stretch sensor, can initiate cellular signaling response in the heart when cardiac preload increases. Previous work demonstrated increased ILK expression could induce angiogenesis to improved heart function after MI. However the patholo-physiological role of ILK in cardiac remodeling after MI is not clear. Method and Results: Hearts were induced to cardiac remodeling by infarction and studied in Sprague-Dawley rats. Until 4 weeks after infarction, ILK expression was increased in non-ischemic tissue in parallel with myocytes hypertrophy and compensatory cardiac function. 8 weeks later, when decompensation of heart function occurred, ILK level returned to baseline. Followed ILK alternation, vascular endothelial growth factor (VEGF) expression and phosphorylation of endothelial nitric oxide synthase (eNOS) was significantly decreased 8 weeks after MI. Histology study also showed significantly microvessel decreased and myocytes loss 8 weeks paralleled with ILK down-regualtion. While ILK expression was maintained by gene delivery, tissue angiogenesis and cardiac function was preserved during cardiac remodeling. Conclusion: Temporally up-regulation of ILK level in non-ischemic myocytes by increased external load is associated with beneficial angiogenesis to maintain infarction-induced cardiac hypertrophy. When ILK expression returns to normal, this cardiac adaptive response for infarction is weaken. Understanding the ILK related mechanism of cardiac maladaptatio

Interplay between cell adhesion and growth factor receptors: from the plasma membrane to the endosomes

The emergence of multicellular animals could only take place once evolution had produced molecular mechanisms for cell adhesion and communication. Today, all metazoans express integrin-type adhesion receptors and receptors for growth factors. Integrins recognize extracellular matrix proteins and respective receptors on other cells and, following ligand binding, can activate the same cellular signaling pathways that are regulated by growth factor receptors. Recent reports have indicated that the two receptor systems also collaborate in many other ways. Here, we review the present information concerning the role of integrins as assisting growth factor receptors and the interplay between the receptors in cell signaling and in the orchestration of receptor recycling

Two Distinct Integrin-Mediated Mechanisms Contribute to Apical Lumen Formation in Epithelial Cells

Background: Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of b1-integrins in epithelial polarization is well established but the significance of the accompanying a-subunits have not been analyzed in detail. Principal Findings: Here we demonstrate that two distinct integrin-dependent pathways regulate formation of apical lumens to ensure robust apical membrane biogenesis under different microenvironmental conditions; 1) a2b1- and a6b4integrins were required to establish a basal cue that depends on Rac1-activity and guides apico-basal cell polarization. 2) a3b1-integrins were implicated in positioning of mitotic spindles in cysts, a process that is essential for Cdc42-driven epithelial hollowing. Significance: Identification of the separate processes driven by particular integrin receptors clarifies the functional hierarchies between the different integrins co-expressed in epithelial cells and provides valuable insight into the complexity of cell-ECM interactions thereby guiding future studies addressing the molecular basis of epithelial morphogenesis durin

PINCH1 Is Transcriptional Regulator in Podocytes That Interacts with WT1 and Represses Podocalyxin Expression

Background: PINCH1, an adaptor protein containing five LIM domains, plays an important role in regulating the integrin-mediated cell adhesion, migration and epithelial-mesenchymal transition. PINCH1 is induced in the fibrotic kidney after injury, and it primarily localizes at the sites of focal adhesion. Whether it can translocate to the nucleus and directly participate in gene regulation is completely unknown. Methodology/Principal Findings: Using cultured glomerular podocytes as a model system, we show that PINCH1 expression was induced by TGF-β1, a fibrogenic cytokine that promotes podocyte dysfunction. Interestingly, increased PINCH1 not only localized at the sites of focal adhesions, but also underwent nuclear translocation after TGF-β1 stimulation. This nuclear translocation of PINCH1 was apparently dependent on the putative nuclear export/localization signals (NES/NLS) at its C-terminus, as deletion or site-directed mutations abolished its nuclear shuttling. Co-immunoprecipitation and pull-down experiments revealed that PINCH1 interacted with Wilms tumor 1 protein (WT1), a nuclear transcription factor that is essential for regulating podocyte-specific gene expression in adult kidney. Interaction of PINCH1 and WT1 was mediated by the LIM1 domain of PINCH1 and C-terminal zinc-finger domain of WT1, which led to the suppression of the WT1-mediated podocalyxin expression in podocytes. PINCH1 also repressed podocalyxin gene transcription in a promoter-luciferase reporter assay. Conclusion/Significance: These results indicate that PINCH1 can shuttle into the nucleus from cytoplasm in podocytes, wherein it interacts with WT1 and suppresses podocyte-specific gene expression. Our studies reveal a previously unrecognized, novel function of PINCH1, in which it acts as a transcriptional regulator through controlling specific gene expression. © 2011 Wang et al

The Small Molecule Inhibitor QLT0267 Radiosensitizes Squamous Cell Carcinoma Cells of the Head and Neck

BACKGROUND: The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC). METHODOLOGY/PRINCIPAL FINDINGS: Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK(floxed/floxed(fl/fl)) and ILK(-/-) mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0-6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; gammaH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK(fl/fl) fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls. CONCLUSIONS/SIGNIFICANCE: Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic

Significance of Thymosin β4 and Implication of PINCH-1-ILK-α-Parvin (PIP) Complex in Human Dilated Cardiomyopathy

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression—the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI

α5β1 Integrin-Mediated Adhesion to Fibronectin Is Required for Axis Elongation and Somitogenesis in Mice

The arginine-glycine-aspartate (RGD) motif in fibronectin (FN) represents the major binding site for α5β1 and αvβ3 integrins. Mice lacking a functional RGD motif in FN (FNRGE/RGE) or α5 integrin develop identical phenotypes characterized by embryonic lethality and a severely shortened posterior trunk with kinked neural tubes. Here we show that the FNRGE/RGE embryos arrest both segmentation and axis elongation. The arrest is evident at about E9.0, corresponding to a stage when gastrulation ceases and the tail bud-derived presomitic mesoderm (PSM) induces α5 integrin expression and assumes axis elongation. At this stage cells of the posterior part of the PSM in wild type embryos are tightly coordinated, express somitic oscillator and cyclic genes required for segmentation, and form a tapered tail bud that extends caudally. In contrast, the posterior PSM cells in FNRGE/RGE embryos lost their tight associations, formed a blunt tail bud unable to extend the body axis, failed to induce the synchronised expression of Notch1 and cyclic genes and cease the formation of new somites. Mechanistically, the interaction of PSM cells with the RGD motif of FN is required for dynamic formation of lamellipodia allowing motility and cell-cell contact formation, as these processes fail when wild type PSM cells are seeded into a FN matrix derived from FNRGE/RGE fibroblasts. Thus, α5β1-mediated adhesion to FN in the PSM regulates the dynamics of membrane protrusions and cell-to-cell communication essential for elongation and segmentation of the body axis

Multiple Roles of Integrin-Linked Kinase in Epidermal Development, Maturation and Pigmentation Revealed by Molecular Profiling

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function

Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation

During Xenopus gastrulation α5β1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the α5β1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the α5β1 integrin through receptor endocytosis